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Diagnosis and management of chronic viral hepatitis:antigens, antibodies and viral genomes.

Stéphane Chevaliez, Jean-Michel Pawlotsky

To cite this version:Stéphane Chevaliez, Jean-Michel Pawlotsky. Diagnosis and management of chronic viral hepatitis:antigens, antibodies and viral genomes.. Baillière’ s Best Practice and Research in Clinical Gastroen-terology, 2008, 22 (6), pp.1031-48. <10.1016/j.bpg.2008.11.004>. <inserm-00368349>

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Diagnosis and management of chronic viral hepatitis: antigens, antibodiesand viral genomesSt phane Chevaliez é 1 2 , Jean-Michel Pawlotsky 1 2 *

IMRB, Institut Mondor de recherche biom dicale 1 é INSERM : U841 , Universit Paris XII Val de Marne é , H pital Henri Mondor 51, av duômal de lattre de tassigny 94010 CRETEIL CEDEX,FR

Centre de r f rence fran ais des h patites B, C et D 2 é é ç é Institut National de la Transfusion Sanguine , FR

* Correspondence should be adressed to: Jean-Michel Pawlotsky <jean-michel.pawlotsky@hmn.aphp.fr >

MESH Keywords Antiviral Agents ; therapeutic use ; Disease Progression ; Genome, Viral ; Hepacivirus ; genetics ; Hepatitis B Antibodies ; blood ; Hepatitis B Antigens ;

blood ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; diagnosis ; drug therapy ; genetics ; Hepatitis C Antibodies ; blood ; Hepatitis C Antigens ; blood ; Hepatitis C,

Chronic ; blood ; diagnosis ; drug therapy ; genetics ; Humans ; Prognosis ; RNA, Viral ; blood

Approximately 500 million people, i.e. one fifth of the world population, are chronically infected with hepatitis B virus (HBV) or

hepatitis C virus (HCV) , . Each year, over 1,5 million people die from HBV- and/or HCV-related chronic liver diseases, such as1 2

end-stage cirrhosis and hepatocellular carcinoma (HCC). HCC is one of the most common cancers worldwide and in 2002, HBV and HCV

have been responsible for at least half a million of these cancers .3

HBV belongs to the family, genus . HBV is the smallest human enveloped DNA virus, with aHepadnaviridae Orthohepadnavirus

genome of approximately 3,200 base pairs. The partially double-stranded circular DNA genome encodes at least four overlapping open

reading frames, including the surface (preS/S), precore/core (preC/C), polymerase (P) and X genes . HBV strains are classified into 84

genotypes (A to H) based-on an 8 or more DNA sequence difference over the full genome , . The different HBV genotypes have% 5 6

distinct geographical distributions and appear to bear different biological properties that may affect the clinical outcome of HBV-related

disease .7 –14

HCV belongs to the family, genus . HCV is an enveloped RNA virus, with a genome of approximately 9,500Flaviviridae Hepacivirus

nucleotides. The single positive-strand RNA genome contains a major open reading frame that encodes a large polyprotein of

approximately 3,000 amino acids. The polyprotein co- and post-translational processing yields a number of structural and non-structural

proteins . HCV strains are classified into 6 major genotypes, and a seventh one has been recently identified , . The HCV15 16 17

genotypes also have distinct geographical distributions and the genotype is an essential prognosis marker of the likelihood of HCV

eradication during antiviral treatment .18

Virological tools are needed to diagnose chronic HBV and HCV infections, they may be useful to establish their prognosis, but they

have found their principal application in guiding treatment decisions and assessing the virological responses to therapy.

VIROLOGICAL TOOLS

The virological diagnosis and monitoring of HBV and HCV infections is based on the use of a variety of virological markers (Table 1

). Six HBV markers are used in clinical practice, including HBs antigen (HBsAg), anti-HBs antibodies, HBe antigen (HBeAg), anti-HBe

antibodies, anti-HBc antibodies (including total anti-HBc antibodies and anti-HBc IgM) and HBV DNA. Three HCV markers are useful in

clinical practice, including total anti-HCV antibodies, HCV genotype and HCV RNA.

Viral antigens and antibodies

Principles of viral antigen and antibody detection and quantification

The detection (and eventually the quantification) of viral antigens and of specific antibodies in body fluids is based on the use of

sandwich enzyme immunoassays (EIAs). Recombinant antigens or antibodies are used to capture circulating antibodies or antigens,

respectively, onto the wells of microtiter plates, microbeads or specific holders adapted to close automated devices. The presence of

antigens or antibodies is revealed by antibodies (in the case of antigen) or anti-antibodies (in the case of antibody) labeled with an enzyme

that catalyzes the transformation of a substrate into a colored compound. The optical density (OD) ratio of the reaction (sample

OD/internal control OD) is proportional to the amount of antigens or antibodies in the sample. EIAs are cheap, easy to use, can be fully

automated and are well adapted to large volume testing.

HBV serological markers

Hepatitis B surface antigen (HBsAg)

Hepatitis B surface antigen is bore by three distinct viral proteins encoded by the pre-S/S gene: the major (or small) surface protein

encoded by the S gene, the most abundantly produced surface protein; the middle surface protein, encoded by the preS2 S gene; and the+large surface protein, encoded by the preS1 preS2 S gene. HBsAg is present in great excess in the blood of HBV-infected patients,+ +

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essentially associated with noninfectious particles including spherical particles and tubular ones (approximately 10 particles per13

milliliter) .19

HBsAg is detected early during acute infection, on average 6 to 10 weeks after exposure and several weeks before the onset of clinical

and biological symptoms ( ). The analytical sensitivity of the available commercial HBsAg EIAs has been improved recently.Figure 1A

Indeed, current HBsAg detection assays detect at least 0.15 nanograms (ng) per milliliter (mL) of circulating HBsAg, i.e. approximately

350 international units (IU) per mL . This improvement has reduced the window period , i.e. the post-exposure period during which20 “ ”HBsAg is undetectable, to up to 9 days . In addition, the ability of HBsAg assays to detect HBV variants bearing nucleotide20

substitutions in the S gene leading to modifications of the tri-dimensional structure of HBsAg has been improved compared to the previous

generations of tests . The specificity of current HBsAg detection assays is of more than 99.5 . False-positive results can be21 –23 %exceptionally observed in pregnant women, autoimmune diseases, and chronic liver diseases of other causes . They can also23 –25

sometimes be observed in heparinized, hemolyzed (hemoglobin above 1.4 g/dL) or icteric (bilirubin above 52.8 mol) blood specimens μ 24

. In practice, it is recommended that the first detection of HBsAg be confirmed by neutralization in order to eliminate a false-positive

result.

Evolution of acute hepatitis B towards chronic HBV carriage is defined by HBsAg persistence for more than 6 months . Under26

certain circumstances, HBsAg may not be detectable during chronic hepatitis B: (i) in low-replication asymptomatic HBV carriers; (ii) in

the case of HBV variants bearing nucleotide substitutions in the S gene leading to the synthesis of an HBsAg that is not recognized by

commercial assays , ; (iii) when infection resolves spontaneously or after successful antiviral therapy in chronic HBV-infected27 28

patients who may subsequently achieve an HBs seroconversion; (iv) in hepatitis delta HBV-co-infected patients, where hepatitis delta virus

most often inhibits HBV replication and expression , .27 29

HBsAg quantification can now be performed by means of a fully automated chemiluminescent microparticle immunoassay. HBsAg

quantification is easy, cheap and may provide a mean to establish the prognosis of antiviral therapy in the future. HBsAg quantification

indeed appears to be a surrogate marker of the amount of covalently closed circular DNA (cccDNA), the persistent intrahepatic form of

HBV DNA, in the hepatocytes and a predictor of a sustained virological response to antiviral treatment off therapy. During antiviral

therapy, HBsAg decrease is a predictor of subsequent HBsAg clearance, the principal goal of antiviral treatment, which may be

subsequently followed by an HBs seroconversion characterized by the appearance of anti-HBs antibodies .30 –32

The estimated annual incidence of spontaneous HBsAg clearance from serum in chronically infected patients is 1 to 2 in% %Caucasians . It may be lower (0.1 to 0.8 ) in high endemicity areas where infection is usually acquired perinatally or in the early33 % %childhood .34 –36

Anti-hepatitis B surface (anti-HBs) antibodies

The presence of anti-HBs antibodies (in the presence of anti-HBc antibodies) witnesses the recovery of chronic HBV infection and

life-long immunity against HBV. The anti-HBs titer often varies over time, and anti-HBs antibodies may become undetectable several

years after the acute episode. The assessment of anti-HBs antibody levels by different assays is not accurate and consistent, yielding

incomparable quantitative results in spite of the calibration with international standards .37

After vaccination against HBV, isolated anti-HBs antibodies must be present at a titer of more than 10 mIU/mL to confer efficient

protection.

Anti-hepatitis B core (anti-HBc) antibodies

Anti-hepatitis B core antibodies are detected early during HBV infection ( ). Two isotypes of anti-HBc antibodies can beFigure 1A

detected, including: anti-HBc IgM, a marker of acute infection that can be also detected at low levels during the immune elimination phase

of chronic hepatitis B and during exacerbations in inactive carriers; anti-HBc IgG which rise in parallel to HBsAg at the acute phase of

infection and persist for life-long whatever the outcome of HBV infection ( ).Table 1

In contrast to anti-HBs antibodies, anti-HBc IgG are not neutralizing . False-negative detection of anti-HBc antibodies mayin vivo

rarely occur in immunosuppressed patients. A rare HBV variant harboring an in-frame deletion of the core gene defective for HBc antigen

synthesis, named HBV-2, has been reported not to elicit a detectable immune response to HBc antigen in immunocompetent patients. We

recently reported the case of an organ donor with low-level HBV DNA (3.0 Log IU/mL) lacking both HBsAg and anti-HBc antibodies;10

several substitutions in both the HBsAg and HBc antigen sequences were found in this donor .38

Anti-HBc antibodies may be the only marker present in chronically infected patients, for instance in low-replication asymptomatic

HBV carriers with low HBsAg production. Attempts to further characterize anti-HBc antibodies in a prognostic purpose, such as

determining anti-HBc IgG subclass profiles or anti-HBc antibody affinity, have failed so far.

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Hepatitis B e antigen (HBeAg) and anti-HBe antibodies

The HBe protein, which bears the HBe antigen, is a nonstructural HBV protein encoded by the preC/C gene. It is secreted by infected

cells under various soluble forms that vary in size between 16 and 20 kDa. The HBe protein contains two epitopes: e1, conformational, and

e2, linear. The e1 epitope sequence overlaps that of the conformational HBc epitope, so that e1 is presented at the surface of HBV

nucleocapsids and expressed, together with HBc, at the surface of infected cells. Although HBe protein secretion does not appear to be

essential for HBV lifecycle, its presence is associated with immune tolerance, high-level HBV replication and high transmissibility.

During acute infection, HBeAg can be detected on average 6 to 12 weeks after exposure ( ). HBeAg clearance is generallyFigure 1A

associated with a decrease of viremia and an aminotransferase flare. It is followed by the appearance of anti-HBe antibodies during the

HBe seroconversion phase. Persistence of HBeAg is generally observed in the patients who develop chronic infection.

In chronic HBV infection, two phenotypes can be observed, including HBeAg-positive and HBeAg-negative chronic hepatitis B. In

HBeAg-positive chronic hepatitis B, the preC/C gene has a wild-type sequence, no anti-HBe antibodies are found, and the presence of

HBeAg in peripheral blood is generally associated with a high HBV DNA level. In HBeAg-negative chronic hepatitis B, the patients are

infected with a variant virus, bearing nucleotide substitutions in the precore region and/or in the basal core promoter region of the preC/C

gene. The most frequent nucleotide substitution occurs at position 1896 (G1896A) in the precore region of the preC/C gene. It inserts a

stop codon in the precore sequence that prevents the HBe protein from being synthesized. The second group of nucleotide polymorphisms

is located within the core promoter region. The most frequent changes are at positions 1762 (A1762T) and 1764 (G1764A). They

down-regulate HBe protein production up to 70 . Other mutations susceptible to alter HBe protein production have also been described % 33

, . Different variants bearing precore and/or basal core promoter mutations can be observed in the same patient in variable proportions.39

Although a few cases of infection with HBeAg-negative viruses have been described in the literature, HBeAg-negative variantsde novo

are generally selected during the immune clearance of chronic HBV infection. An important outcome of the immunoelimination phase is

indeed seroconversion from HBeAg to anti-HBe, characterized by the clearance of HBeAg followed by the appearance of anti-HBe

antibodies. The seroconversion phase may result in an HBeAg-negative chronic hepatitis B or in an inactive carrier state characterized by

normal aminotransferase levels and undetectable or low-level (< 2 000 IU/mL) HBV DNA. The estimated annual incidence of spontaneous

HBeAg seroclearence in chronically infected patients is 5 to 15 , .% % 40 41

HCV serological markers

Total anti-HCV antibodies

The serological window, characterized by detectable HCV RNA and core antigen in the absence of anti-HCV antibodies, has been

estimated to be of approximately 60 days on average . Anti-HCV antibodies appear on average 2 to 8 weeks after the acute phase of42

infection and persist for life in patients who develop chronic HCV infection ( ). The presence of both anti-HCV antibodies andFigure 1B

HCV RNA does not allow to distinguish acute hepatitis C from an acute exacerbation of chronic hepatitis C or an acute hepatitis of another

cause in a patient with chronic hepatitis C. The anti-HCV IgG avidity index within the first 8 days following the onset of clinical

symptoms may be useful in identifying actual acute HCV infection cases .43

The significance of the presence of anti-HCV IgM during HCV infection is unclear. Anti-HCV IgM have been reported in 50 to 93% %of patients with acute hepatitis C and 50 to 70 of patients with chronic hepatitis C . Therefore, anti-HCV IgM cannot be used as% % 44 –46

a reliable marker of acute HCV infection, and IgM assays have not be used in clinical practice. However, the serial measurements of the

anti-HCV IgM titers based on at least three determinations from the fifth to the fifteenth day from the onset of the symptoms may identify

patients with acute hepatitis C .47

Serological determination of HCV genotype

The HCV genotype can be determined by means of a competitive ELISA assay using genotype-specific antigens . This assay48

allows to identify the 6 HCV genotypes (1 to 6) but not the subtype.

Viral genome

Genome detection and quantification

Principles of genome detection and quantification

Viral genome detection and quantification can be achieved by using two categories of molecular biology-based techniques, including

target amplification (such as (PCR)) and signal amplification (such as hybrid capture or the branched DNApolymerase chain reaction

assay). Whatever technique used, HBV DNA and HCV RNA international units per milliliter must be preferred to any other quantitative

unit and should now be implemented in all commercial quantitative assays. Conversion factors can be used to establish a relationship

between the IUs and the non standardized copies , . The classical techniques for viral genome detection and quantification are now49 50

progressively being replaced by real-time PCR assays in most virology laboratories ( ).Table 2

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Real-time PCR techniques have a broad dynamic range of quantification, well suited to the clinical needs (upper range of

quantification: 7 to 8 Log IU/mL). Real-time PCR is more sensitive than classical PCR, with lower limits of detection of the order of 1010

to 15 IU/mL for both HBV DNA and HCV RNA. Real-time PCR assays do not yield false-positive results due to carryover

contaminations, and they can be fully automated. Real-time PCR has become the technique of choice to detect and quantify viral genomes

in clinical practice. Several real-time PCR assays are now commercially available for the detection and quantification of both HBV DNA

and HCV RNA ( ).Table 2

HBV DNA detection and quantification

HBV DNA detection and quantification is useful in clinical practice to: (i) to diagnose chronic hepatitis B with viral replication; (ii)

establish the prognosis of liver disease and assess the risk of progression towards cirrhosis and hepatocellular carcinoma ; (iii) identify51

patients who need antiviral therapy and offer them the most adapted treatment; (iv) monitor the virological response to therapy and identify

amino acid substitutions responsible for resistance to nucleos(t)ide analogues.

The presence of HBV DNA in peripheral blood is a reliable marker of HBV replication. HBV DNA is detectable within a few days

after infection. It generally increases to reach a peak at the time of acute hepatitis, and then progressively decreases and disappears when

the infection resolves spontaneously . In the patients progressing towards chronic HBsAg carriage, chronic infection evolves through52

successive phases. HBV DNA levels are not stable over time and depend on the infection phase: the immunotolerance phase is

characterized by high levels of viral replication; the immuno-elimination phase is characterized by generally lower, often fluctuating HBV

DNA levels; the inactive carrier stage is characterized by very low or undetectable levels of viral replication, depending on the sensitivity

of the assay; reactivation phases, that are facilitated by immunosuppressive therapies, are generally associated with high levels of

replication.

HBV DNA assays based on real-time PCR technology are now replacing the classical techniques in most virology laboratories. Three

real-time PCR platforms are currently available for detection and quantification of HBV DNA: the Cobas Taqman platform, which can ®

be used together with automated sample preparation with the Cobas AmpliPrep system (CAP-CTM, Roche Molecular System,®

Pleasanton, California), the Real-Art HBV PCR Assay (Artus-Biotech, Qiagen, Hamburg, Germany), and the Abbott platform (Abbott ®

Diagnostic, Chicago Illinois), which uses the 2000 amplification platform together with the 2000 device for sample preparation (m RT m SP

). These assays have been shown to accurately quantify HBV DNA levels in clinical practice , .Table 2 53 54

HCV RNA detection and quantification

HCV RNA detection and quantification is useful in clinical practice to: (i) diagnose chronic HCV infection; (ii) identify patients who

need antiviral therapy; (iii) monitor the virological responses to antiviral therapy, and (iv) in the future, identify amino acid substitutions

responsible for resistance to specific inhibitors of HCV viral proteins .50

The presence of HCV RNA in peripheral blood is a reliable marker of HCV replication. HCV RNA can be detected 1 to 3 weeks after

infection, approximately 1 month before the appearance of total anti-HCV antibodies. The presence of HCV RNA after 6 months signs

chronic HCV infection. HCV RNA levels remain relatively stable over time in chronically infected patients.

HCV RNA assays based on real time PCR are now used in clinical virology laboratories for RNA detection and quantification. Two

real-time PCR platforms are currently available for detection and quantification of HCV RNA: the Cobas Taqman platform, which can®be used together with automated sample preparation with the Cobas AmpliPrep system (CAP-CTM, Roche Molecular System), and the®Abbott platform (Abbott Diagnostic), which uses the 2000 amplification platform together with the 2000 device for samplem RT m SP

preparation. Another assay, developed by Siemens Medical Solutions Diagnostics (Tarrytown, New-York) will be available soon (Table 2

). The intrinsic performances of available tests differ. Indeed, approximately 15 of HCV genotype 2 and 30 of HCV genotype 4% %samples are substantially underestimated in the CAP-CTM, most likely because of nucleotide mismatches, whereas this problem has not

been found with the Abbott assay , .55 56

Analysis of the nucleotide sequence of the viral genome

Principles of nucleotide sequence analysis

Viral genome sequence analysis is generally based on direct sequencing (so-called population sequencing) that provides the full

sequence of the analyzed fragment, or reverse hybridization that identifies specific nucleotides or motifs at given positions. In practice,

signature sequences are used: (i) to classify viral strains into phylogenetic groups of clinical interest, such as the 8 HBV genotype (A to H)

or the 6 HCV genotypes (1 to 6); (ii) to identify nucleotide substitutions of the precore and basal core promoter regions of the preC/C

gene; (iii) to identify amino acid substitutions known to confer viral resistance to specific antiviral inhibitors.

Genotype determination

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The reference method for viral genotype determination is phylogenetic analysis of sequences generated after PCR amplification of a

portion of the viral genome relative to reference sequences. Commercial assays have been developed. The Trugene HBV Genotyping Kit ®

(Siemens Medical Solutions Diagnostics) can determine the HBV genotype after amplification of a region overlapping the open reading

frame encoding the S and P genes. The Trugene 5 NC HCV Genotyping kit (Siemens Medical Solutions Diagnostics) has been developed ® ′for HCV genotype determination by direct sequencing of a portion of the 5 non-coding region (NCR) of the viral genome. Direct sequence′analysis is the gold standard for genomic sequence analysis. However, it only identifies viral variants representing at least 20 to 25 of the%circulating viral populations.

Reverse hybridization of PCR amplicons to membrane-bound probes is more sensitive than direct sequence analysis to detect minor

variants representing as few as 5 the whole viral population . Line probe assays (INNO-LiPA, Innogenetics, Gent, Belgium) use a% 57

series of short immobilized oligonucleotide probes to discriminate among different PCR fragments. A commercial assays has been

developed for HBV genotype determination (INNO-LiPA HBV Genotyping) . The most recent version of the line probe assay for HCV58

genotype determination (VERSANT HCV Genotype 2.0 Assay, Siemens Medical Solutions Diagnostics) bears consistently improved ®

accuracy for HCV genotype 1 subtype and HCV genotype 6 determination compared to the previous assays .59 –62

The utility of HBV genotype determination in clinical practice is debated. The positive and negative predictive values of the HBV

genotype on disease progression and treatment outcomes have not been determined at the individual patient level, and further large clinical

studies are warranted before implementing HBV genotype determination in practice . In contrast, HCV genotype determination is63

mandated before the initiation of therapy with pegylated interferon alpha and ribavirin because it is the best predictor of treatment response

and it determines the dose of ribavirin and treatment duration .18

Identification of amino acid substitutions associated with viral resistance

Direct sequence analysis and reverse hybridization methods are used to identify amino acid substitutions known to confer resistance to

antiviral drugs before the viral level increases. At present, early detection of HBV substitutions conferring resistance to nucleos(t)ide

analogues is used to alter therapy in order to avoid rebound and hepatitis flare. Commercial assays are available. The Trugene HBV ®

Genotyping Kit (Siemens Medical Solutions Diagnotics) is based on direct sequence analysis of a portion of the reverse transcriptase

domain of the HBV polymerase gene . A new generation of reverse hybridization assay, INNO-LiPA HBV DR v3, has been developed64

in 2008 to detect amino acid substitutions associated with lamivudine, adefovir and entecavir resistance. Reverse hybridization methods

have been shown to detect changes in viral quasispecies composition early, on average 6 months before direct sequencing analysis , .65 66

PRACTICAL USE OF VIROLOGICAL TOOLS IN THE MANAGEMENT OF CHRONICHEPATITIS B

Serological and molecular HBV markers are used in clinical practice to diagnose chronic HBV infection, assess the prognosis of the

disease, guide therapy and monitor treatment responses.

Diagnosis of chronic HBV infection

The persistence of HBsAg for more than 6 months defines chronic HBV carriage. In a chronic HBV carrier, chronic hepatitis B is

defined by an elevated serum HBV DNA (generally > 2 10 to 2 10 IU/mL), with persistent or intermittent elevation of× 3 × 4

aminotransferase levels and signs of chronic hepatitis B on liver biopsy . In a chronic HBV carrier, the HBV DNA level should be67 –70

systematically measured by means of a sensitive and accurate method. Real-time PCR assays should be preferred and the results must be

expressed in international units per milliliter . The HBV DNA level is indeed a key determinant of liver disease progression towards26

cirrhosis or HCC , . HBV DNA level measurement is also crucial for therapeutic decision-making.51 71

shows the kinetics of HBV markers during chronic HBV infection. HBsAg and total anti-HBc antibodies are alwaysFigure 1A

present. The presence of HBeAg (in the absence of anti-HBe antibodies) is generally associated with high-level viral replication and high

transmissibility. HBV replication levels are substantially lower on average in HBeAg-negative patients. Inactive HBV carriers have a low

level of viral replication (< 2 10 IU/mL), normal aminotransferase levels, no HBeAg and positive anti-HBe antibodies.× 3

Assessment of disease severity and prognosis

The HBV DNA level and aminotransferase activity provide valuable prognostic information. The REVEAL study, which included

more than 3,000 Taiwanese patients with chronic HBV infection, showed that an HBV DNA level higher than 4.3 Log (i.e. 2 1010 × 4

IU/mL) was associated with a significant risk of progression of chronic hepatitis B to cirrhosis and HCC, independently of the HBeAg

serostatus and level of aminotransferase activity , . In addition, the risk of HCC is significantly related to the level of HBV51 71

replication and starts raising above 2 10 IU/mL . The risk of HCC is low in the absence of detectable HBV DNA, except in patients× 3 72

with cirrhosis.

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Treatment of HBV infection

The goal of chronic hepatitis B therapy is to prevent progression of liver disease to its life-threatening complications, cirrhosis and

HCC. This can be achieved if HBV replication is durably abolished or significantly reduced. In HBeAg-positive patients, HBeAg

clearance followed by the HBe seroconversion phase can be achieved in some cases with short-term therapy and ensures long-term control

of viral replication. In HBeAg-negative patients, long-term antiviral suppression of viral replication is needed in most cases. The loss of

HBsAg, eventually associated with an HBs seroconversion, is the most desirable endpoint of therapy but is rarely achieved.

Decision to treat

The decision to treat chronic hepatitis B is based on the assessment of multiple parameters including clinical, biological and histologic

parameters. Antiviral treatment is currently recommended in patients with an HBV DNA titer above 2,000 IU/mL, elevated serum alanine

aminotransferase activity (above the upper limit of normal), and/or evidence of chronic hepatitis with or without cirrhosis. Antiviral

treatment should be considered in patients with a low level of viral replication who show mild to moderate necro-inflammatory activity

and/or fibrosis. In HBV-infected patients with normal aminotransferase activity and an HBV DNA level below 2,000 IU/mL, repeated

HBV DNA and ALT determinations are recommended every 3 to 6 months.

Selection of optimal therapy

The current treatment of chronic hepatitis B is based on the use of two categories of antiviral compounds: pegylated interferon alpha

and nucleos(t)ide analogues that inhibit the reverse transcriptase domain of viral polymerase. Five nucleos(t)ide analogues are approved in

Europe or in US for the treatment of chronic hepatitis B, including lamivudine, adefovir dipivoxil, entecavir, telbivudine and tenofovir

disoproxil fumarate.

In HBeAg-positive patients, first-line treatment with pegylated interferon alpha is recommended if aminotransferase levels are

elevated and the HBV DNA level is moderate (below 2 10 IU/mL). Although HBV genotypes A and B globally appear to better respond× 6

to interferon-based therapy than genotypes C and D, HBV genotype determination is not yet recommended to guide the therapeutic

decision in the absence of strong individual predictive value.

Most of the other patients, whatever the HBe serostatus, and in HBeAg-positive patients who did not achieve an HBe seroconversion

during or after interferon alpha therapy, the use of nucleos(t)ide analogues is recommended. Entecavir or tenofovir must be preferred as

first-line treatment because they both potently inhibit HBV replication and they have a high genetic barrier to resistance. Combinations of

nucleos(t)ide analogues with no cross-resistance increases the genetic barrier to resistance and thus better prevents resistance on the

long-term than mono- or sequential therapy. De novo combinations can be used as first-line treatment in patients with high HBV DNA

levels and are unlikely to reach undetectable HBV DNA on monotherapy.

Treatment monitoring

HBV treatment monitoring is based on HBV DNA quantifications and ALT determinations every 3 to 6 months, whatever the HBe

serostatus and antiviral treatment .63

In HBeAg-positive patients, treatment efficacy is witness by the loss of HBeAg with may be followed by a seroconversion to anti-HBe

antibodies. It is generally associated with a profound reduction of serum HBV DNA levels and normalization of aminotransferase activity.

Ideally in HBe seroconverters, HBV DNA should be undetectable with a sensitive real-time PCR assay (lower limit of detection of the

order 10 to 15 IU/mL) and aminotransferase activity should be normal . If this is not the case, the patient may have switched from73

HBeAg-positive to HBeAg-negative therapy and may need sustained therapeutic suppression of viral replication.

In HBeAg-positive patients with no HBe seroconversion and in HBeAg-negative patients, the goal of antiviral treatment is to achieve a

profound and durable HBV DNA suppression. The HBV DNA level should be undetectable on treatment with a sensitive real-time PCR

assay (lower limit of detection: 10 to 15 IU/mL) . If the HBV DNA level remains detectable after 48 weeks, a second antiviral63

compound with no cross-resistance with the first one must be added in order to prevent subsequent resistance.

In the patients who have responded and have been compliant, resistance should be suspected if the HBV DNA level rises by more than

1 Log IU/mL above nadir in two consecutive samples taken one month part , . The identification of selected amino acid10 63 74

substitutions known to be associated with resistance to the administered drug(s) by means of molecular testing can help guide treatment

adaptation. Consensus decisional algorithms will need to be established before systematic genotypic resistance testing can be

recommended to adapt the treatment strategy to the resistance profile of the infecting viral strain.

Monitoring of untreated chronic HBV infections

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Antiviral treatment is not recommended in patients with an HBV DNA level below 2,000 IU/mL and normal aminonotransferase

activity . However, a regular monitoring should be performed. Aminotransferase activity should be assessed every month for the three67

first months, and then every 6 12 months. In untreated patients, the severity of liver inflammation and fibrosis must be evaluated by means–of a liver biopsy or non-invasive serological or ultrasound-based testing when persistent or intermittent elevation of aminotransferase

levels has been observed. HBV DNA quantification every 6 months is useful to detect an increase in viral replication and eventually

reconsider the treatment indication.

PRACTICAL USE OF VIROLOGICAL TOOLS IN THE MANAGEMENT OF CHRONICHEPATITIS C

Serological and molecular markers are used in clinical practice to diagnose chronic hepatitis C, guide treatment decisions and monitor

the antiviral efficacy of treatment.

Diagnosis of chronic HCV infection

The persistence of HCV RNA for more than 6 months defines chronic HCV infection. In patients with clinical and/or biological signs

of chronic liver disease, chronic hepatitis C is diagnosed by the simultaneous presence of anti-HCV antibodies and HCV RNA. Detectable

HCV replication in the absence of anti-HCV antibodies is exceptional with current anti-HCV enzyme immunoassays. It is almost

exclusively observed in profoundly immunodepressed, hemodialysis or agamaglobulinemic subjects , .75 76

Treatment of HCV infection

The current standard treatment for chronic hepatitis C is the combination of pegylated interferon alpha-2a or -2b and ribavirin . The77

efficacy endpoint of chronic hepatitis C treatment is the sustained virological response (SVR), defined by an undetectable HCV RNA in

serum with a sensitive assay (lower limit of detection of 50 IU/mL or less) 24 weeks after the end of treatment.

Decision to treat and indication of treatment

The decision to treat chronic hepatitis C depends on multiple parameters including a precise assessment of the severity of liver disease,

the presence of absolute or relative contra-indications to therapy, and the patient s willingness to be treated.’

HCV genotype determination should be systematically determined before treatment, as it determines the indication, the duration of

treatment, the dose of ribavirin, and the virological monitoring procedure ( ) . Genotypes 2- and 3-infected patients require 24Figure 2 18

weeks of treatment and a low dose of ribavirin, i.e. 800 mg daily. In contrast, genotype 1-, 4-, 5- and 6-infected patients require 48 weeks

of treatment and a high, body-weight based dose of ribavirin, i.e. 1000 to 1400 mg daily.

Treatment monitoring

Monitoring of HCV RNA levels is recommended to tailor treatment to the actual virological response. A sensitive assay with a brioad

range of quantification should be used. A real-time PCR assay should ideally be used. In HCV genotype 1-infected patients, the HCV

RNA level should be measured before therapy and 12 weeks after its initiation ( ). The lack of a 12-week virological response (i.e.Figure 2

no change or an HCV RNA decrease of less than 2 Log at week 12) indicates that the patient has virtually no chance to achieve an SVR10

and should stop treatment. In contrast, treatment must be continued when a 2 Log drop in HCV RNA level has been observed at week 12,10

until week 48 if HCV RNA is undetectable, until week 72 if HCV RNA is still detectable at week 12 , .28 78

Recent studies have suggested that the patients who achive a rapid virological response, defined by an undetectable HCV RNA (< 50

IU/mL) at week 4 of therapy, could benefit from shorter treatment duration, i.e. 24 weeks in patients infected with HCV genotypes 1, 4, 5

or 6 and 12 to 16 weeks in those infected with HCV genotypes 2 or 3 . . These results however need confirmation and new79 –83 84

algorithms should be drawn to tailor treatment duration to the virological response at week 4 without loosing a chance of viral eradication.

The sustained virological response corresponds to a cure of infection in more than 99 of cases. In the mid-term future, triple%combination therapy with pegylated interferon alpha, ribavirin and a specific HCV inhibitor will likely become the standard treatment of

chronic hepatitis C. The SVR will remain the endpoint of therapy. On-treatment monitoring and the corresponding decision algorithms

will need to be established.

Monitoring of untreated chronic HCV infections

In patients with no indication or with contra-indications to therapy, the HCV RNA level has no prognostic value. The level of HCV

replication does not correlate with the severity of liver disease, nor with the risk of liver disease progression to cirrhosis or HCC. Repeated

aminotransferase level assessments are recommended. Assessment of liver inflammation and fibrosis by means of liver biopsy or

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Page /8 13

non-invasive serological or ultrasound-based testing is needed in the case of persistent or intermittent elevatiosn of aminotransferase levels

.77

References: 1 . Lavanchy D . Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures . J Viral Hepat . 2004 ; 11 : 97 - 107 2 . Shepard CW , Finelli L , Alter MJ . Global epidemiology of hepatitis C virus infection . Lancet Infect Dis . 2005 ; 5 : 558 - 67 3 . Parkin DM . The global health burden of infection-associated cancers in the year 2002 . Int J Cancer . 2006 ; 118 : 3030 - 44 4 . Robinson WS , Clayton DA , Greenman RL . DNA of a human hepatitis B virus candidate . J Virol . 1974 ; 14 : 384 - 91 5 . Kidd-Ljunggren K , Miyakawa Y , Kidd AH . Genetic variability in hepatitis B viruses . J Gen Virol . 2002 ; 83 : 1267 - 80 6 . Schaefer S . Hepatitis B virus taxonomy and hepatitis B virus genotypes . World J Gastroenterol . 2007 ; 13 : 14 - 21 7 . Chu CM , Liaw YF . Genotype C hepatitis B virus infection is associated with a higher risk of reactivation of hepatitis B and progression to cirrhosis than genotype B: a

longitudinal study of hepatitis B e antigen-positive patients with normal aminotransferase levels at baseline . J Hepatol . 2005 ; 43 : 411 - 7 8 . Chu CM , Liaw YF . Predictive factors for reactivation of hepatitis B following hepatitis B e antigen seroconversion in chronic hepatitis B . Gastroenterology . 2007 ; 133 :

1458 - 65 9 . Flink HJ , van Zonneveld M , Hansen BE , de Man RA , Schalm SW , Janssen HL . Treatment with Peg-interferon alpha-2b for HBeAg-positive chronic hepatitis B: HBsAg

loss is associated with HBV genotype . Am J Gastroenterol . 2006 ; 101 : 297 - 303 10 . Funk ML , Rosenberg DM , Lok AS . Worldwide epidemiology of HBeAg-negative chronic hepatitis B and associated precore and core promoter variants . J Viral Hepat .

2002 ; 9 : 52 - 61 11 . Grandjacques C , Pradat P , Stuyver L , Chevallier M , Chevallier P , Pichoud C , Maisonnas M , Trepo C , Zoulim F . Rapid detection of genotypes and mutations in the

pre-core promoter and the pre-core region of hepatitis B virus genome: correlation with viral persistence and disease severity . J Hepatol . 2000 ; 33 : 430 - 9 12 . Janssen HL , van Zonneveld M , Senturk H , Zeuzem S , Akarca US , Cakaloglu Y , Simon C , So TM , Gerken G , de Man RA , Niesters HG , Zondervan P , Hansen B ,

Schalm SW . Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial . Lancet . 2005 ; 365 : 123 - 9 13 . Livingston SE , Simonetti JP , McMahon BJ , Bulkow LR , Hurlburt KJ , Homan CE , Snowball MM , Cagle HH , Williams JL , Chulanov VP . Hepatitis B virus

genotypes in Alaska Native people with hepatocellular carcinoma: preponderance of genotype F . J Infect Dis . 2007 ; 195 : 5 - 11 14 . Orito E , Ichida T , Sakugawa H , Sata M , Horiike N , Hino K , Okita K , Okanoue T , Iino S , Tanaka E , Suzuki K , Watanabe H , Hige S , Mizokami M . Geographic

distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan . Hepatology . 2001 ; 34 : 590 - 4 15 . Chevaliez S , Pawlotsky JM . Editor: Tan S-L . HCV genome and life cycle . Hepatitis C viruses: genome and molecular biology: Horizon Bioscience . 2006 ; 5 - 47 16 . Simmonds P , Bukh J , Combet C , Deleage G , Enomoto N , Feinstone S , Halfon P , Inchauspe G , Kuiken C , Maertens G , Mizokami M , Murphy DG , Okamoto H ,

Pawlotsky JM , Penin F , Sablon E , Shin IT , Stuyver LJ , Thiel HJ , Viazov S , Weiner AJ , Widell A . Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes . Hepatology . 2005 ; 42 : 962 - 73

17 . Murphy DG , Chamberland J , Dandavino R , Sablon E . A new genotype of hepatitis C virus originating from central Africa . Hepatology . 2007 ; 46 : (Suppl 1 ) 623A - 18 . Hadziyannis SJ , Sette H Jr , Morgan TR , Balan V , Diago M , Marcellin P , Ramadori G , Bodenheimer H Jr , Bernstein D , Rizzetto M , Zeuzem S , Pockros PJ , Lin A ,

Ackrill AM . Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose . Ann Intern Med . 2004 ; 140 : 346 - 55

19 . Kan M , Gerlich W . Structure and molecular virology . Blackwell ; 2005 ; 20 . Biswas R , Tabor E , Hsia CC , Wright DJ , Laycock ME , Fiebig EW , Peddada L , Smith R , Schreiber GB , Epstein JS , Nemo GJ , Busch MP . Comparative sensitivity

of HBV NATs and HBsAg assays for detection of acute HBV infection . Transfusion . 2003 ; 43 : 788 - 98 21 . Gibb R , Nimmo GR , O Loughlin ’ P , Lowe P , Drummond D . Detection of HBsAg mutants in a population with a low prevalence of hepatitis B virus infection . J Med

Virol . 2007 ; 79 : 351 - 5 22 . Muhlbacher A , Weber B , Burgisser P , Eiras A , Cabrera J , Louisirirotchanakul S , Tiller FW , Kim HS , Helden JV , Bossi V , Echevarria JM . Multicenter study of a

new fully automated HBsAg screening assay with enhanced sensitivity for the detection of HBV mutants . Med Microbiol Immunol . 2008 ; 197 : 55 - 64 23 . Weber B , Van der Taelem-Brule N , Berger A , Simon F , Geudin M , Ritter J . Evaluation of a new automated assay for hepatitis B surface antigen (HBsAg) detection

VIDAS HBsAg Ultra . J Virol Methods . 2006 ; 135 : 109 - 17 24 . Weber B , Bayer A , Kirch P , Schluter V , Schlieper D , Melchior W . Improved detection of hepatitis B virus surface antigen by a new rapid automated assay . J Clin

Microbiol . 1999 ; 37 : 2639 - 47 25 . Weber B , Dengler T , Berger A , Doerr HW , Rabenau H . Evaluation of two new automated assays for hepatitis B virus surface antigen (HBsAg) detection: IMMULITE

HBsAg and IMMULITE 2000 HBsAg . J Clin Microbiol . 2003 ; 41 : 135 - 43 26 . Lok AS , McMahon BJ . Chronic hepatitis B . Hepatology . 2001 ; 34 : 1225 - 41 27 . Shukla NB , Poles MA . Hepatitis B virus infection: co-infection with hepatitis C virus, hepatitis D virus, and human immunodeficiency virus . Clin Liver Dis . 2004 ; 8 : 445 - 60

28 . Berg T , von Wagner M , Nasser S , Sarrazin C , Heintges T , Gerlach T , Buggisch P , Goeser T , Rasenack J , Pape GR , Schmidt WE , Kallinowski B , Klinker H , Spengler U , Martus P , Alshuth U , Zeuzem S . Extended treatment duration for hepatitis C virus type 1: comparing 48 versus 72 weeks of peginterferon-alfa-2a plus ribavirin .

Gastroenterology . 2006 ; 130 : 1086 - 97 29 . Su CW , Huang YH , Huo TI , Shih HH , Sheen IJ , Chen SW , Lee PC , Lee SD , Wu JC . Genotypes and viremia of hepatitis B and D viruses are associated with

outcomes of chronic hepatitis D patients . Gastroenterology . 2006 ; 130 : 1625 - 35 30 . Akhan SC , Yulugkural Z , Vahaboglu H . Response to interferon-alpha in chronic hepatitis B patients with and without precore mutant strain and effects on HBsAg titers .

Chemotherapy . 2007 ; 53 : 402 - 6 31 . Chan HL , Wong VW , Tse AM , Tse CH , Chim AM , Chan HY , Wong GL , Sung JJ . Serum hepatitis B surface antigen quantitation can reflect hepatitis B virus in the

liver and predict treatment response . Clin Gastroenterol Hepatol . 2007 ; 5 : 1462 - 8 32 . Ozaras R , Tabak F , Tahan V , Ozturk R , Akin H , Mert A , Senturk H . Correlation of quantitative assay of HBsAg and HBV DNA levels during chronic HBV treatment

. Dig Dis Sci . 2008 ; 33 . Fattovich G , Bortolotti F , Donato F . Natural history of chronic hepatitis B: special emphasis on disease progression and prognostic factors . J Hepatol . 2008 ; 48 : 335 -

52 34 . Liaw YF , Sheen IS , Chen TJ , Chu CM , Pao CC . Incidence, determinants and significance of delayed clearance of serum HBsAg in chronic hepatitis B virus infection: a

prospective study . Hepatology . 1991 ; 13 : 627 - 31 35 . Nam SW , Jung JJ , Bae SH , Choi JY , Yoon SK , Cho SH , Han JY , Han NI , Yang JM , Lee YS . Clinical outcomes of delayed clearance of serum HBsAg in patients

with chronic HBV infection . Korean J Intern Med . 2007 ; 22 : 73 - 6 36 . Wu TT , Hsu HC , Chen DS , Sheu JC , Su IJ , Chen SL , Chuang SM . Clearance of hepatitis B surface antigen (HBsAg) after surgical resection of hepatocellular

carcinoma . J Hepatol . 1987 ; 4 : 45 - 51 37 . Huzly D , Schenk T , Jilg W , Neumann-Haefelin D . Comparison of nine commercially available assays for quantification of antibody response to hepatitis B virus surface

antigen . J Clin Microbiol . 2008 ; 46 : 1298 - 306 38 . Challine D , Chevaliez S , Pawlotsky JM . Hepatitis B virus (HBV) replication in organ, tissue and cell donors with and without HBV serological markers .

Gastroenterology . 2008 ; in press 39 . Ingman M , Lindqvist B , Kidd-Ljunggren K . Novel mutation in hepatitis B virus preventing HBeAg production and resembling primate strains . J Gen Virol . 2006 ; 87 : 307 - 10

Best Pract Res Clin Gastroenterol . Author manuscript

Page /9 13

40 . Brown SD , Barbara AJ , Lambert T , Wilson DV . Spontaneous loss of HBeAg and development of anti-HBe during long-term follow-up of blood donors found to be HBsAg-positive . Br J Biomed Sci . 1995 ; 52 : 106 - 9

41 . Lok AS , Heathcote EJ , Hoofnagle JH . Management of hepatitis B: 2000-summary of a workshop . Gastroenterology . 2001 ; 120 : 1828 - 53 42 . Busch MP , Glynn SA , Stramer SL , Strong DM , Caglioti S , Wright DJ , Pappalardo B , Kleinman SH . A new strategy for estimating risks of transfusion-transmitted

viral infections based on rates of detection of recently infected donors . Transfusion . 2005 ; 45 : 254 - 64 43 . Coppola N , Pisapia R , Marrocco C , Martini S , Vatiero LM , Messina V , Tonziello G , Sagnelli C , Filippini P , Piccinino F , Sagnelli E . Anti-HCV IgG avidity index

in acute hepatitis C . J Clin Virol . 2007 ; 40 : 110 - 5 44 . Hellstrom UB , Sylvan SP , Decker RH , Sonnerborg A . Immunoglobulin M reactivity towards the immunologically active region sp75 of the core protein of hepatitis C

virus (HCV) in chronic HCV infection . J Med Virol . 1993 ; 39 : 325 - 32 45 . Negro F , Troonen H , Michel G , Giostra E , Albrecht M , Perrin L , Hadengue A . Lack of monomeric IgM anti-hepatitis C virus (HCV) core antibodies in patients with

chronic HCV infection . J Virol Methods . 1996 ; 60 : 179 - 82 46 . Sagnelli E , Coppola N , Marrocco C , Coviello G , Rossi G , Battaglia M , Sagnelli C , Messina V , Tonziello A , Scolastico C , Filippini P . Diagnosis of HCV related

acute hepatitis by serial determination of IgM to HCV: a preliminary observation . J Biol Regul Homeost Agents . 2003 ; 17 : 207 - 10 47 . Sagnelli E , Coppola N , Marrocco C , Coviello G , Battaglia M , Messina V , Rossi G , Sagnelli C , Scolastico C , Filippini P . Diagnosis of hepatitis C virus related acute

hepatitis by serial determination of IgM anti-HCV titres . J Hepatol . 2005 ; 42 : 646 - 51 48 . Pawlotsky JM , Prescott L , Simmonds P , Pellet C , Laurent-Puig P , Labonne C , Darthuy F , Remire J , Duval J , Buffet C , Etienne JP , Dhumeaux D , Dussaix E .

Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay . J Clin Microbiol . 1997 ; 35 : 1734 - 9 49 . Gish RG , Locarnini SA . Chronic hepatitis B: current testing strategies . Clin Gastroenterol Hepatol . 2006 ; 4 : 666 - 76 50 . Pawlotsky JM . Use and interpretation of virological tests for hepatitis C . Hepatology . 2002 ; 36 : S65 - 73 51 . Chen CJ , Yang HI , Su J , Jen CL , You SL , Lu SN , Huang GT , Iloeje UH . Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus

DNA level . JAMA . 2006 ; 295 : 65 - 73 52 . Whalley SA , Murray JM , Brown D , Webster GJ , Emery VC , Dusheiko GM , Perelson AS . Kinetics of acute hepatitis B virus infection in humans . J Exp Med . 2001 ; 193 : 847 - 54 53 . Chevaliez S , Bouvier-Alias M , Laperche S , Pawlotsky JM . Performance of the Cobas AmpliPrep/Cobas TaqMan real-time PCR assay for hepatitis B virus DNA

quantification . J Clin Microbiol . 2008 ; 46 : 1716 - 23 54 . Thibault V , Pichoud C , Mullen C , Rhoads J , Smith JB , Bitbol A , Thamm S , Zoulim F . Characterization of a new sensitive PCR assay for quantification of viral DNA

isolated from patients with hepatitis B virus infections . J Clin Microbiol . 2007 ; 45 : 3948 - 53 55 . Chevaliez S , Bouvier-Alias M , Brillet R , Pawlotsky JM . Overestimation and underestimation of hepatitis C virus RNA levels in a widely used real-time polymerase

chain reaction-based method . Hepatology . 2007 ; 46 : 22 - 31 56 . Chevaliez S , Bouvier-Alias M , Pawlotsky JM . Performance of the Abbott m2000SP/m2000RT real-time polymerase chain reaction assay for hepatitis C virus RNA

quantification . submitted 57 . Sertoz RY , Erensoy S , Pas S , Akarca US , Ozgenc F , Yamazhan T , Ozacar T , Niesters HG . Comparison of sequence analysis and INNO-LiPA HBV DR line probe

assay in patients with chronic hepatitis B . J Chemother . 2005 ; 17 : 514 - 20 58 . Hussain M , Chu CJ , Sablon E , Lok AS . Rapid and sensitive assays for determination of hepatitis B virus (HBV) genotypes and detection of HBV precore and core

promoter variants . J Clin Microbiol . 2003 ; 41 : 3699 - 705 59 . Bouchardeau F , Cantaloube JF , Chevaliez S , Portal C , Razer A , Lefrere JJ , Pawlotsky JM , De Micco P , Laperche S . Improvement of hepatitis C virus (HCV)

genotype determination with the new version of the INNO-LiPA HCV assay . J Clin Microbiol . 2007 ; 45 : 1140 - 5 60 . Chevaliez S , Bouvier-Alias M , Vandervenet C , Pawlotsky JM . HCV genotype determination in clinical practice: weaknesses of assays based on the 5 noncoding region′

and improvement with the core-coding region . Hepatology . 2007 ; 46 : (Suppl1 ) 839A - 61 . Stelzl E , van der Meer C , Gouw R , Beld M , Grahovac M , Marth E , Kessler HH . Determination of the hepatitis C virus subtype: comparison of sequencing and reverse

hybridization assays . Clin Chem Lab Med . 2007 ; 45 : 167 - 70 62 . Verbeeck J , Stanley MJ , Shieh J , Celis L , Huyck E , Wollants E , Morimoto J , Farrior A , Sablon E , Jankowski-Hennig M , Schaper C , Johnson P , Van Ranst M , Van

Brussel M . Evaluation of Versant hepatitis C virus genotype assay (LiPA) 2.0 . J Clin Microbiol . 2008 ; 46 : 1901 - 6 63 . Pawlotsky JM , Dusheiko G , Hatzakis A , Lau D , Lau G , Liang TJ , Locarnini S , Martin P , Richman DD , Zoulim F . Virologic monitoring of hepatitis B virus therapy

in clinical trials and practice: recommendations for a standardized approach . Gastroenterology . 2008 ; 134 : 405 - 15 64 . Gintowt AA , Germer JJ , Mitchell PS , Yao JD . Evaluation of the MagNA Pure LC used with the Trugene HBV Genotyping Kit . J Clin Virol . 2005 ; 34 : 155 - 7 65 . Libbrecht E , Doutreloigne J , Van De Velde H , Yuen MF , Lai CL , Shapiro F , Sablon E . Evolution of primary and compensatory lamivudine resistance mutations in

chronic hepatitis B virus-infected patients during long-term lamivudine treatment, assessed by a line probe assay . J Clin Microbiol . 2007 ; 45 : 3935 - 41 66 . Osiowy C , Villeneuve JP , Heathcote EJ , Giles E , Borlang J . Detection of rtN236T and rtA181V/T mutations associated with resistance to adefovir dipivoxil in samples

from patients with chronic hepatitis B virus infection by the INNO-LiPA HBV DR line probe assay (version 2) . J Clin Microbiol . 2006 ; 44 : 1994 - 7 67 . Keeffe EB , Dieterich DT , Han SH , Jacobson IM , Martin P , Schiff ER , Tobias H , Wright TL . A treatment algorithm for the management of chronic hepatitis B virus

infection in the United States: an update . Clin Gastroenterol Hepatol . 2006 ; 4 : 936 - 62 68 . Lok AS . Chronic hepatitis B . N Engl J Med . 2002 ; 346 : 1682 - 3 69 . Lok AS , McMahon BJ . Chronic hepatitis B: update of recommendations . Hepatology . 2004 ; 39 : 857 - 61 70 . Prati D , Taioli E , Zanella A , Della Torre E , Butelli S , Del Vecchio E , Vianello L , Zanuso F , Mozzi F , Milani S , Conte D , Colombo M , Sirchia G . Updated

definitions of healthy ranges for serum alanine aminotransferase levels . Ann Intern Med . 2002 ; 137 : 1 - 10 71 . Iloeje UH , Yang HI , Su J , Jen CL , You SL , Chen CJ . Predicting cirrhosis risk based on the level of circulating hepatitis B viral load . Gastroenterology . 2006 ; 130 : 678 - 86

72 . Chen CJ , Yang HI , Su J , Jen CL , You SL , Chen CF , Iloeje UH . Serial monitoring of viral load and serum alanine transferase level and the risk of hepatocellular carcinoma (HCC): R.E.V.E.A.L. -HBV study update . J Hepatol . 2008 ; 48 : (Suppl 2 ) S61 -

73 . Lai CL , Yuen MF . The natural history and treatment of chronic hepatitis B: a critical evaluation of standard treatment criteria and end points . Ann Intern Med . 2007 ; 147 : 58 - 61

74 . Lok AS , McMahon BJ . Chronic hepatitis B . Hepatology . 2007 ; 45 : 507 - 39 75 . Lok AS , Chien D , Choo QL , Chan TM , Chiu EK , Cheng IK , Houghton M , Kuo G . Antibody response to core, envelope and nonstructural hepatitis C virus antigens:

comparison of immunocompetent and immunosuppressed patients . Hepatology . 1993 ; 18 : 497 - 502 76 . Thio CL , Nolt KR , Astemborski J , Vlahov D , Nelson KE , Thomas DL . Screening for hepatitis C virus in human immunodeficiency virus-infected individuals . J Clin

Microbiol . 2000 ; 38 : 575 - 7 77 . NIH Consensus Statement on Management of Hepatitis C . NIH Consens State Sci Statements . 2002 ; 19 : 1 - 46 78 . Sanchez-Tapias JM , Diago M , Escartin P , Enriquez J , Romero-Gomez M , Barcena R , Crespo J , Andrade R , Martinez-Bauer E , Perez R , Testillano M , Planas R ,

Sola R , Garcia-Bengoechea M , Garcia-Samaniego J , Munoz-Sanchez M , Moreno-Otero R . Peginterferon-alfa2a plus ribavirin for 48 versus 72 weeks in patients with detectable hepatitis C virus RNA at week 4 of treatment . Gastroenterology . 2006 ; 131 : 451 - 60

79 . Dalgard O , Bjoro K , Hellum KB , Myrvang B , Ritland S , Skaug K , Raknerud N , Bell H . Treatment with pegylated interferon and ribavarin in HCV infection with genotype 2 or 3 for 14 weeks: a pilot study . Hepatology . 2004 ; 40 : 1260 - 5

80 . Ferenci P , Bergholz U , Laferl H , Scherzer TM , Maieron A , Gschwantler M , Brunner H , Hubmann RR , Datz C , Bishof M , Stauber R , Steindl-Munda P . 24 weeks treatment regimen with peginterferon alpha-2a (40 kDa) (Pegasys) plus ribavirin (Copegus) in HCV genotype 1 or 4 super-responders “ ” . J Hepatol . 2006 ; 44 : S6 -

Best Pract Res Clin Gastroenterol . Author manuscript

Page /10 13

81 . Mangia A , Santoro R , Minerva N , Ricci GL , Carretta V , Persico M , Vinelli F , Scotto G , Bacca D , Annese M , Romano M , Zechini F , Sogari F , Spirito F , Andriulli A . Peginterferon alfa-2b and ribavirin for 12 vs. 24 weeks in HCV genotype 2 or 3 . N Engl J Med . 2005 ; 352 : 2609 - 17

82 . von Wagner M , Huber M , Berg T , Hinrichsen H , Rasenack J , Heintges T , Bergk A , Bernsmeier C , Haussinger D , Herrmann E , Zeuzem S . Peginterferon-alpha-2a (40KD) and ribavirin for 16 or 24 weeks in patients with genotype 2 or 3 chronic hepatitis C . Gastroenterology . 2005 ; 129 : 522 - 7

83 . Zeuzem S , Buti M , Ferenci P , Sperl J , Horsmans Y , Cianciara J , Ibranyi E , Weiland O , Noviello S , Brass C , Albrecht J . Efficacy of 24 weeks treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C infected with genotype 1 and low pretreatment viremia . J Hepatol . 2006 ; 44 : 97 - 103

84 . Fried MW , Hadziyannis SJ , Shiffman M , Messinger D , Zeuzem S . Rapid virological response is a more important predictor of sustained virological response (SVR) than genotype in patients with chronic hepatitis C virus infection . J Hepatol . 2008 ; 48 : (Suppl 2 ) S5 -

Figure 1Kinetics of virological markers during chronic viral hepatitis: (A) chronic HBV infection; (C) chronic HCV infection.

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Figure 2Algorithms for the use of HCV virologicals tools in the treatment of chronic hepatitis C, according to the HCV genotype.

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Table 1Virological markers for the diagnosis and management of chronic HBV and HCV infections

Hepatitis B virus

Direct markers

Hepatitis B surface antigen (HBsAg)Hepatitis B e antigen (HBeAg)HBV DNA

HBV genotype*

HBV resistance substitutions*

ccDNA*

Indirect markers

Anti-HBs antibodiesAnti-HBc antibodies (IgM & IgG)Anti-HBe antibodies

Hepatitis C virus

Direct markers

Capsid antigen*

HCV RNAHCV genotypeIndirect markers

Total anti-HCV antibodies * not routinely available

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Table 2Commercially available real-time PCR assays for HBV DNA and HCV RNA detection and quantification

Assay Manufacturer MethodLower limit of

detection Dynamic range

Hepatitis B virus

Cobas Taqman HBV Roche Molecular Systems, Pleasanton,California

Real-time PCR on Cobas Taqman after automated extraction (CobasAmpliprep)

12 IU/mL 54 IU/mL -110 000 000 IU/mL

Real-Art HBV PCRassay

Qiagen, Hamburg, Germany Real-time PCR after manual extraction 4 IU/mL 4 to 9 IU/mL -100 000 000IU/mL

Abbott Real-time HBV Abbott Diagnostic, Chicago, Illinois Real-time PCR on 2000 after automated extraction ( 2000 )m RT m SP 10 IU/mL 10 UI/mL - 1 000 000 000IU/mL

Hepatitis C virus

Cobas Taqman HCV Roche Molecular Systems, Pleasanton,California

Real-time PCR on Cobas Taqman after automated extraction (CobasAmpliprep)

15 IU/mL 46 IU/mL - 69 000 000 IU/mL

Abbott Real-time HCV Abbott Diagnostic, Chicago, Illinois Real-time PCR 2000 after automated extraction( 2000 )m RT m SP 12 to 30 IU/mL 12 IU/mL -100 000 000 UI/mL